6-羟基多巴胺通过CaMKII调节Cav1.3对大鼠黑质多巴胺能神经元的损伤研究

Effect of 6-hydroxydopamine on substantia nigra dopaminergic neuron injury in rats by CaMKII regulating Cav1.3

目的探讨Ca2+/钙调蛋白依赖的蛋白激酶II(CaMKII)和Cav1.3在6-羟基多巴胺处理大鼠原代黑质多巴胺能神经元损伤中的作用。方法取出生24 h内大鼠黑质多巴胺能神经元进行原代培养,12d后分别进行加药处理。使用免疫印迹法、免疫共沉淀法检测6-羟基多巴胺对神经元CaMKII活性和Cav1.3表达情况、Cav1.3-CaMKII复合体形成情况。采用MTT法、siRNA敲减技术检测6-羟基多巴胺对大鼠原代多巴胺神经元细胞死亡情况的影响。结果 6-羟基多巴胺处理组Cav1.3表达水平和p-CaMKII水平明显高于对照组,两组比较差异具有统计学意义(P<0.05)。6-羟基多巴胺处理组Cav1.3-CaMKII复合体水平明显高于对照组,两组比较差异具有统计学意义(P<0.05)。6-羟基多巴胺处理组p-CaMKII和Cav1.3水平显著高于对照组(P<0.05),而KN能明显抑制6-羟基多巴胺引起的p-CaMKII和Cav1.3增加,两组比较差异具有统计学意义(P<0.05)。SiCav1.3能抑制50%以上Cav1.3表达,抑制Cav1.3和CaMKII,改善6-羟基多巴胺引起的细胞损伤,两组比较差异具有统计学意义(P<0.05)。结论 6-羟基多巴胺处理大鼠原代多巴胺神经元引起的细胞损伤与Cav1.3和CaMKII表达和活性增加相关,CaMKII通过与Cav1.3形成复合体来直接调节Cav1.3的功能,抑制Cav1.3和Ca MKII可改善6-羟基多巴胺导致的细胞损伤。

Objective To investigate the effect of 6-hydroxydopamine on substantia nigra dopaminergic neuron injury in rats by CaMKII regulating Cav1.3. Methods Dopaminergic neurons in substantia nigra of rats within 24 h of birth were cultured in primary culture, and treated with drugs for 12 d. Effects of 6-hydroxydopamine on CaMKII activity, Cav1.3 expression, Cav1.3-CaMKII complex formation, and 6-hydroxydopamine on cell death of primary dopamine neurons in rats were detected by Western blotting method and co-immunoprecipitation method. MTT method and siRNA knockout technique were used to detect the effect of 6-hydroxydopamine on the death of primary dopamine neurons in rats. Results The expression level of Cav1.3 and p-CaMKII in the 6-hydroxydopamine group were significantly higher than those in the control group, and there were differences between two groups(P < 0.05). The level of Cav1.3-CaMKII complex in the 6-hydroxydopamine group was significantly higher than that in the control group, and there were differences between two groups(P < 0.05). The levels of p-CaMKII and Cav1.3 in the 6-hydroxydopamine group were significantly higher than those in the control group, while KN significantly inhibited the increase of p-CaMKII and Cav1.3 induced by 6-hydroxydopamine, and there were differences between two groups(P < 0.05). SiCav1.3 inhibited Cav1.3 expression by more than 50%, inhibited Cav1.3 and CaMKII, and improved cell damage caused by 6-hydroxydopamine, and there were differences between two groups(P < 0.05). Conclusion Cell damage induced by 6-hydroxydopamine of primary dopamine neurons in rats is associated with increasing expression and activity of Cav1.3 and CaMKII. CaMKII directly regulates Cav1.3 function by forming a complex with Cav1.3, and inhibiting Cav1.3 and CaMKII can improve cell damage induced by 6-hydroxydopamine.